The primary aim of this proposal is to characterize the thermodynamics of specific interactions between the heme group and the surrounding protein matrix in hemoglobin. This parameterization should provide some of the fundamental information necessary to evaluate current models of hemoglobin allostery. This primary goal includes several components. First hemoglobin derivatives must be prepared for which specific thermodynamic and structural effects (e.g. metal imidazole bond strength, heme stereochemistry) can be evaluated. We will therefore prepare a series of metal substituted Hbs, using techniques developed during the initial grant period. These include: Fe(III)Hb, Zn(II)Hb, Mn(III)Hb, Fe alpha2Zn beta2, Fe alpha2 Mn beta2, Mn alpha2 Fe beta2, Cu(II)Hb, Fe alpha2 Cu beta2, Cu alpha2 Fe beta2, RuCOHb, IrCOHb, Zn alpha2 Fe beta2. Other hybrids may be prepared based on the results obtained with this group. Once purified, the quaternary structures of these substituted hemoglobins will be evaluated, using three primary techniques: high resolution (400 MHz) NMR, U.V. (280-290 nm) circular dichroism, and subunit dissociation measurements. An attempt will be made to relate the observed 4 degree structure to specific features of the heme site, including heme stereochemistry, coordination number, and metalimidazole bond strength. Enthalpies of metal-imidazole binding in simple porphyrins will be independently determined. Such characterization will represent a major advance in the chemistry of metal substituted hemoglobins. Finally, we will attempt to characterize the allosteric equilibrium constant, L equals (T) over (R) for these derivatives. Three independent approaches have been devised for their characterization. These include preparation and sickling measurements on metal substituted Hbs.